The mechanism(s) of breast cancer progression to steroid receptor (SR)-positive but hormone refractory breast cancer remain elusive. Much of the research in this field is focused on the linkage of estrogen receptor-? (ER) to signal transduction, as mediated by peptide growth factor activation of tyrosine kinase receptors; these molecules are important clinical targets. However, progesterone receptors (PR) may also play a key role as mediators of early breast cancer progression. Recently, we uncovered a novel mechanism of hormone-independent hyperactivation of PR transcriptional activity that is mediated by membrane-initiated phosphorylation events. In response to mitogenic protein kinases (c-Src, MAPKs, CDK2) often elevated in breast cancer, persistent phosphorylation of PR Ser294 blocks ligand-induced sumoylation at K388 (a repressive modification). Abundant total PR is a marker of good tumor behavior. However, we hypothesize that de-repressed phospho-PR acts on genes whose products mediate early breast cancer progression or bad tumor behavior. One check on PR hyperactivity includes rapid ligand-dependent degradation by the ubiquitin-proteasome pathway. Indeed, in the presence of hormone, phospho-PR undergoes rapid turnover, often rendering the low abundance protein undetectable by standard antibody-binding assays (i.e. clinically used IHC). Clinical observations of PR loss have led to the incorrect conclusion that PR transcriptional activity is unimportant in PR-low breast cancers. However, our studies reveal that phospho-PR is transcriptionally hyperactive and remarkably, targets genes that are not sensitive to progesterone or progestins; IRS-1 is an example of a ligand-independent phospho-PR gene. Furthermore, under conditions of high kinase activities, Ser294-phosphorylated PRs are not sumoylated, and thus fail to transrepress ER but may instead cooperate with ER at non-classical (non-PRE containing) gene targets in the complete absence of steroid hormones; STC-1 is an example of an ER-gene that is de-repressed by phospho-PR. Notably, the proliferation of breast cancer cell models expressing mutant PRs that cannot be sumoylated (K388R) is very sensitive to estrogen, but resistant to anti-estrogen. Similarly, we predict that phospho-PR signaling drives the growth of some hormone-refractory breast cancers that can be identified by a unique PR gene signature. Herein, we will define the phospho-PR gene signature using unique cell line models engineered for inducible PR expression. The role of the PR sumoylation/phosphorylation switch in the regulation of hormone responsiveness at endogenous genes will be defined (Mechanisms; Aim 1). Our unique phospho-PR gene signature will be validated in in vitro models of tam- and AI- (aromatase inhibitor) resistance and in human tumors (Validation; Aim 2). Finally, our innovative hypothesis will be tested in AIB1-transgenic mice, an in vivo model of SR-driven (ER+/PR+) and tam-resistant breast cancer (Biology; Aim 3). Little is known about how PR and ER/PR interactions contribute to hormone resistance in breast tumors. Our studies on phospho- PR signaling will provide valuable insight into the coordinated regulation of PR and ER function by pathways that can be easily targeted for therapeutic intervention. PUBLIC HEALTH RELEVANCE: These studies are focused on progesterone receptor (PR) action as it relates to mechanisms of hormone refractory breast cancer progression and therapeutic targeting. Our goal is to show that growth factors de-repress PR and thereby alter PR function and interaction with ER at endogenous genes important for breast cancer cell proliferation, survival, and early progression to endocrine resistance. A unique phospho-PR gene signature will be defined in breast cancer cell lines engineered to express versions of PR representing known post-translational modifications, validated in well-characterized independent models of tamoxifen or aromatase inhibitor-resistant breast cancer (expressing endogenous wt PR), and extended into both in vitro and in vivo translational models of steroid receptor-positive endocrine-resistance.